Michal Obořil

Physiological and proteomic approaches to evaluate the role of sterol binding in elicitin-induced resistance

Cryptogein is a proteinaceous elicitor secreted by Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modelling experiments, by site-directed mutagenesis a series of cryptogein variants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded well with the previously reported structural data. Induction of the synthesis of reactive oxygen species (ROS) in tobacco cells in suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant proteins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defence genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the ω-loop, might not be principal factors in either ROS production or resistance induction. Nevertheless, the results support the importance of the ω-loop for the interaction of the protein with the high affinity binding site on the plasma membrane.

Elicitin–membrane interaction is driven by a positive charge on the protein surface: Role of Lys13 residue in lipids loading and resistance induction

Elicitins are family of small proteins secreted by species of the pathogenic fungus Phytophthora inducing a defence reaction in plants. They contain a hydrophobic cavity capable of binding sterols and fatty acids, and on the basis of their pI they are classified as either α-elicitins or more necrotising β-elicitins. The residue Lys13 was previously identified as a key determinant of the necrotising activity of basic elicitins. In the present study we describe changes in the ability of cryptogein, a β-elicitin inducing a hypersensitive response in tobacco, to transfer sterols and fatty acids between micelles and liposomes upon Lys13Val mutation. We propose that the change in activity is influenced by the elimination of positive charge on the surface of cryptogein, which is significant for correct positioning of the protein during lipid loading, without adversely affecting the binding of sterol to the cavity of the protein. Compared to wild type cryptogein, mutation Lys13Val resulted in lowered expression of defence-related genes and compromised resistance to Phytophthora parasitica. Furthermore, resistance induced by Lys13Val mutant was similar to that induced by acidic elicitin capsicein containing at amino position 13 valine Determined results sustained a crucial role of positive lysine residues on the surface of basic elicitins and suggested their significant role in correct protein-membrane interaction and thus on their biological activity.

Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein–Protein Interactions Influenced by Selected Lysine Residues

Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6–7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1–3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

The effect of cryptogein with changed abilities to transfer sterols and altered charge distribution on extracellular alkalinization, ROS and NO generation, lipid peroxidation and LOX gene transcription in Nicotiana tabacum

Cryptogein, a protein from oomycete Phytophthora cryptogea, induces a hypersensitive cell death in Nicotiana tabacum. We prepared a new series of cryptogein mutant proteins with altered abilities to bind sterols and with altered charge distribution in the proteins. The effect of the mutations on the cryptogein ability to induce plant defence mechanisms associated with hypersensitive cell death were examined. Our results with new mutants support the previous findings that the sterol binding does not influence synthesis of ROS, cytosol acidification and development of leaf necrosis as these events seem to be more likely affected by the charge distribution and the overall protein structure. This hypothesis was also applicable on other mechanisms involved in the execution of plant cell death such as the NO generation, the stimulation of lipid peroxidation (determination of malondialdehyde and hydroxy fatty acids levels) and LOX gene transcription. In addition, the ability to bind sterols was found to serve not only for pathogen utilisation in its own metabolism but also to have an important function for the destabilization of plant membrane facilitating the pathogen spread inside the plant tissue as well as intensively contributing to the development of plant cell death. Considering the insertion of charged amino acid residues in the protein structure, the change localized in the protein surface affected its biological activity more effectively than that change inside the protein cavity. Moreover, the insertion of negative charged amino acids influenced mainly the events involved in the early phase of defence reaction, while the positive residues affected especially the necrotic activity of cryptogein.

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

In the published online version, the affiliations were mixed up. Corrected affiliation section is shown below. Also, the update has also been reflected in the author group section above.The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.